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Original Article
Institute for Life Sciences, Kitasato University, Tokyo and 2Oriental Medicine Research Center, The Kitasato
Institute, Tokyo, Japan
Herbal formulas of traditional Japanese (Kampo), Chinese and Korean medicines usually comprise
multiple herbs in a single formula. These medicines are expected to show their clinical effects by
chemical, pharmacological and pharmaceutical combination effects of multi-herbs. However, little
effort has been made so far to scientifically clarify the nature of such combination effects. Interestingly, for example, though a Kampo medicine Juzen-taiho-to (Shi-Quan-Da-Bu-Tang in Chinese)
stimulates the immune functions of Peyers patch cells, none of its single component herbs shows such
activity. We thus examined the combination effect of herbs in the Juzen-taiho-to formula for the
expression of its immuno-stimulating activity. Juzen-taiho-to, a composite formula of 10 herbs, has
been generally considered to comprise two kinds of basic formula, each of which consists of four different herbs in addition to two others. The combinations of herbs based on these two basic formulas
were evaluated for their stimulating activities on cytokine production from murine Peyers patch cells
both in vitro and ex vivo. Combined decoction of six among 10 herbs in Juzen-taiho-to is crucial for
the expression of its stimulating activity on Peyers patch cells. 3D-HPLC analysis of the ingredients in
the fractions from the combined decoctions indicated that, in addition to quantitative changes of
ingredients, alterations occur in their chemical composition by decoction of different herbs. The stimulating activity of Juzen-taiho-to on Peyers patch cells results from the combination effect of its six
essential component herbs. This combination effect is based on physicochemical interactions among
the ingredients of the component herbs.
Keywords: combination effect lignincarbohydrate complexes Peyers patch cells
Introduction
A number of herbal formulas of traditional Japanese medicines (Kampo), and Chinese and Korean ones as well, are
characterized by the use of mixtures of several herbs (multiherbs) in a single formula. These multi-herb formulas are
usually prepared in various dosage forms such as decocted
extracts, pills, powders, tablets etc. in a traditional way.
Therefore, particularly in the case of decocted extracts with
boiling water, one has to expect the possibility that some
chemical interactions take place among natural constituents
existing in the component herbs of the formula during decoction. Decoction may change the extraction rates of the active
ingredients and/or produce new artificial substances, which
For reprints and all correspondence: Haruki Yamada, Kitasato Institute for
Life Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
E-mail: yamada@lisci.kitasato-u.ac.jp
84
Juzen-taiho-to
SMT
SKT
alone
with ASR
with CC
alone
with ASR
with CC
Rehmanniae Radix (3 g)
Paeoniae Radix (3 g)
Cnidii Rhizoma (3 g)
Angelicae Radix (3 g)
Ginseng Radix (3 g)
Poria (3 g)
Glycyrrhizae Radix (3 g)
*
*
*
*
Asterisks indicate presence of herb in the formulation listed at the top of each column.
Full activity.
No activity.
Statistically significant activity.
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85
Results
The fractions (F-3) containing lignincarbohydrate complexes, which were prepared from Juzen-taiho-to (TJ-48),
and F-3-related fractions from hot water extracts of each of
the 10 single herbs of TJ-48, were compared for their modulatory activity on the intestinal immune system. Although F-3
obtained from TJ-48 exhibited a potent activity, none of the
F-3-related fractions from each single component herb had
such activity (Fig. 1). When F-3-related fractions of the 10
single herbs were mixed in equal amounts, or in ratios according to their yields, and were measured for activity, these
mixed F-3-related fractions also failed to express any activity
(Fig. 2A). These results clearly indicate that the combined
decoction of over two kinds of single herbs in the formula of
Juzen-taiho-to is necessary for obtaining F-3 possessing
activity. To further analyze this interesting phenomenon,
identification of the essential combination is necessary. How-
86
Figure 1. Intestinal immune system modulating activity of lignincarbohydrate complexes containing fractions (F-3) prepared from Juzen-taiho-to (TJ-48) and
F-3-related fractions from component herbs. Filled squares, 10 g/ml; open squares, 100 g/ml.
Figure 2. (A) Intestinal immune system modulating activity of combined F-3-related fractions, which were prepared by mixing of F-3-related fractions
obtained from each of the 10 component herbs in equal ratios (a) and in ratios according to their yields (b). (B) Intestinal immune system modulating activity
of various F-3-related fractions prepared from decoctions of SMT or SKT with or without ASR or CC, and from the decoction of the combination of SMT and
SKT. Filled squares, 10 g/ml; open squares, 100 g/ml.
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87
Figure 3. (A) Intestinal immune system modulating activity of various F-3-related fractions prepared from decoctions of SKT with ASR alone or with ASR and
CC combined, and from decoction of the combination of ASR and CC. (B) Effect of omitting each component herb from the combination of SKT with ASR
on intestinal immune system modulating activity of their F-3-related fractions. Filled squares, 10 g/ml; open squares, 100 g/ml. *P < 0.05 versus control.
N.S., not significant.
Figure 4. Ex vivo intestinal immune system modulating activity of F-3related fraction prepared from SKT or SKT with ASR and CC.
88
Figure 5. Gel filtration patterns on Sephadex G-25 of F-3-related fractions prepared from the formulation of SKT (A) or the combination of SKT with ASR
and CC (B). Closed circles, carbohydrate (490 nm); open circles, uronic acid (520 nm); closed triangles, phenolics or protein (280 nm).
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89
Figure 6. (A) Intestinal immune system modulating activity of subfractions of F-3-related fractions prepared from the formulation of SKT alone or from the
combination of SKT with ASR and CC. Filled squares, 10 g/ml; open squares, 100 g/ml. *P < 0.05 versus control. (B) Dose-dependent intestinal immune
system modulating activity of F-3-1 from Fig. 5. Open circles, F-3-1 from SKT; filled squares, F-3-1 from SKT with ASR and CC. *P < 0.005 versus data of F3-1 from SKT.
Table 2. Property of subfractions obtained from F-3 of the prescriptions of SKT, SKT with ASR and CC and TJ-48
SKT
TJ-48
F-3-1-1A
F-3-1
F-3-2
F-3-3
F-3-4
F-3-1
F-3-2
F-3-3
F-3-4
Yield (%)
9.2
9.2
61.1
20.4
7.6
4.2
38.8
49.4
79.2
84.9
n.d.
127.8
57.0
54.9
Arabinose
8.6
9.0
16.5
Rhamnose
3.5
2.9
7.0
Fucose
0.02
0.06
1.8
Xylose
1.8
2.1
4.2
Glucuronic acid
9.2
3.6
3.4
Galacturonic acid
7.7
4.8
11.5
Mannose
4.0
8.0
5.8
Galactose
8.0
6.0
15.6
Glucose
57.3
63.2
32.4
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Figure 7. HPLC patterns of F-3-4 prepared from SKT with ASR and CC (A, C) and from SKT alone (B, D). (A, B) Monitored at 250 nm; (C, D) Monitored
at 280 nm. The peaks with the arrows were significantly different between both F-3-4.
Discussion
In pharmacological study of the formulas in Kampo medicine, previous investigators have noted interesting phenomena when several single herbs are combined to be decocted
into one formula. For example, Hosoya has indicated that
antitussive action of the hot water extract of Ephedra Herba
is enhanced by the combined decoction of this herb with
three other kinds of single herbs, such as Armeniacae Semen,
Glycyrrhizae Radix and Gypsum Fibrosum, while each of its
hot water extracts alone has no antitussive action (1). It was
also found that the duration and strength of antitussive effect
of Ephedra Herba were prolonged and potentiated by this
combined decoction compared with Ephedra Herba alone.
As another example, a dried extract from one formula of
Kampo medicine, Sho-saiko-to-go-Keishi-ka-shakuyaku-to
(TJ-960), which consists of 10 kinds of single herbs, shows
anti-convulsant action on seizures induced by the administration of pentetrazole to guinea pigs. However, if any single
herb except Zingiberis Rhizoma from the multi-herbs of this
formula is omitted, the anti-convulsant activity of the hot
water extract from the complete formula disappears, suggesting that the combined decoction of nine single herbs in the
formula of TJ-960 is necessary for expression of the activity
(1). However, what constitutes the chemical, pharmacological and pharmaceutical basis for such phenomena of combination effects remains obscure.
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Acknowledgments
Part of the present work was supported by a fund from
Tsumura & Co., Tokyo. Part of the present work was also
supported by The 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan. We thank Ms F. Honma and Ms E.
Yoshida for their technical assistance.
91
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Received October 23, 2003; accepted January 19, 2004